\(\textit {Vp}\)StyA1/\(\textit {Vp}\)StyA2B of \(\textit {variovorax paradoxus}\) EPS
- Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein \(\textit {(Vp}\)StyA1) and a two domain protein \(\textit {(Vp}\)StyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as \(\textit {Vp}\)StyA1/\(\textit {Vp}\)StyA2B of \(\textit {Variovorax paradoxus}\) EPS. \(\textit {Vp}\)StyA2B serves mainly as NADH:FAD-oxidoreductase. A \(K_{m}\) of 33.6 \(\pm\) 4.0 \(\mu\)M for FAD and a \(k_{cat}\) of 22.3\(\pm\) 1.1 \(s^{−1}\) were determined and resulted in a catalytic efficiency (\(k_{cat} K_ {m}^{−1}\)) of 0.64 \(s^{−1} \mu M^{−1}\). To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (\(k_{cat} K_{m}^{−1} of 5.21 s^{−1} \mu M^{−1}\)) while keeping wildtype NADH-affinity and -oxidation activity. Both components, \(\textit {Vp}\)StyA2B and \(\textit {Vp}\)StyA1, showed monooxygenase activity on styrene of 0.14 U \(mg^{−1}\) and 0.46 U \(mg^{−1}\), as well as on benzyl methyl sulfide of 1.62 U \(mg^{−1}\) and 3.11 U \(mg^{−1}\), respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. \(\textit {Vp}\)StyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly (\(\it S\))-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of \(\textit {Vp}\)StyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.
Author: | Dirk TischlerORCiDGND, Ringo SchwabeGND, Lucas SiegelGND, Kristin JoffroyGND, Stefan R. KaschabekGND, Anika ScholtissekGND, Thomas HeineORCiDGND |
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URN: | urn:nbn:de:hbz:294-67296 |
DOI: | https://doi.org/10.3390/molecules23040809 |
Parent Title (English): | Molecules |
Subtitle (English): | an aryl alkyl sulfoxidase rather than a styrene epoxidizing monooxygenase |
Publisher: | MDPI |
Place of publication: | Basel |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2019/11/14 |
Date of first Publication: | 2018/04/02 |
Publishing Institution: | Ruhr-Universität Bochum, Universitätsbibliothek |
Tag: | enantioselective biotransformation; epoxidation; flavoprotein; fusion protein; protein linker; soil microorganism; sulfoxidation; two-component monooxygenase |
Volume: | 23 |
Issue: | 4, Article 809 |
First Page: | 809-1 |
Last Page: | 809-13 |
Institutes/Facilities: | Nachwuchsgruppe Mikrobielle Biotechnologie |
open_access (DINI-Set): | open_access |
faculties: | Fakultät für Biologie und Biotechnologie |
Licence (English): | Creative Commons - CC BY 4.0 - Attribution 4.0 International |